A multi-glycomic platform for the analysis of food carbohydrates - Monosaccharide, linkage and polysaccharide (FITDOG) composition analyses of different varieties of apple (figure 5)

Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome, and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity, and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis where the analyte might be any food item or digested product. The carbohydrates are extracted by… and the resulting samples are subjected to employing rapid-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides, and alcohol soluble carbohydrates are performed in 96-well plate format to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS, and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan-microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome, and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health.

碳水化合物是多数膳食中占比最高的营养素，对健康具有深远影响。诸如单糖与淀粉之类的碳水化合物组分可提供能量，而不可消化的组分即被视为膳食纤维，可抵达结肠，为构成肠道微生物群（gut microbiota）的数十万亿微生物提供营养底物。膳食碳水化合物、胃肠道、肠道微生物组（gut microbiome）与宿主健康之间的相互作用，由其分子结构所决定。然而，当前用于食品聚糖分析的方法，在灵敏度、特异性与通量上均无法满足定量解析这类多样结构的需求。本方案详述了一种用于食品碳水化合物分析的多糖组学分析方法，其分析物可为任意食品样品或消化产物。碳水化合物经提取后，所得样品将采用高通量液相色谱-串联质谱（LC-MS/MS）技术进行分析。实验采用96孔板体系完成单糖、糖苷键、多糖及醇溶性碳水化合物的定量分析，以减少所需样品的生物量并提升分析通量。本方案详细提供了样品前处理、LC-MS/MS分析及数据分析的分步操作流程。我们通过一系列多样化食品、不同苹果品种及多种发酵食品的实例，展示了本方案的应用效果。此外，我们还验证了该方法在解析无菌小鼠与定植小鼠体内聚糖-微生物相互作用中的实用性。该方法体系为解析膳食纤维、肠道微生物组与人体生理机能之间的关联提供了研究框架。相关结构信息将进一步为营养学与临床喂养研究提供指导，从而加深我们对膳食在营养与健康中作用的认知。